BaF3 cell transfection protocol

One Nucleofection® Sample contains:

2 x 106 cells
2 µg plasmid DNA
100 µl Cell Line Nucleofector® Solution V

1.1 Please make sure that the entire supplement is added to the Nucleofector® Solution
1.2 Prepare 12-well plates by filling appropriate number of wells with 1 ml of supplemented culture media and pre-incubate/equilibrate plates in a humidified 37°C/5% CO2 incubator
1.3 Count an aliquot of the cells and determine cell density
1.4 Centrifuge the required number of cells (2 x 106 cells per sample) at 90xg for 10 minutes at room temperature. Remove supernatant completely
1.5 Re-suspend the cell pellet carefully in 100 µl room-temperature Nucleofector® Solution per sample
Note Avoid leaving the cells in Nucleofector® Solution for extended periods of time (longer than 15 minutes), as this may reduce cell viability and gene transfer efficiency.
1.6 Combine 100 µl of cell suspension with 2 µg DNA,
1.7 Transfer cell/DNA suspension into certified cuvette (sample must cover the bottom of the cuvette without air bubbles). Close the cuvette with the cap
1.8 Select the appropriate Nucleofector® Program X-001(X-01 for Nucleofector® I Device)
1.9 Insert the cuvette with cell/DNA suspension into the Nucleofector® Cuvette Holder and apply the selected program by pressing the X-button
1.10 Take the cuvette out of the holder once the program is finished
1.11 Immediately add ~500 µl of the pre-equilibrated culture medium to the cuvette and gently transfer the sample into the prepared 12-well plate (final volume 1.5 ml media per well). Use the supplied pipettes and avoid repeated aspiration of the sample

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